The studies in the previous years has led to a hypothesis that the state of one region of the structures of proteins is coupled in a specific way with those of distant regions after folding of some unit of the amino acid sequence to generate extra force for stabilization of the structure. To investigate the role of individual residues in this hypothetical coupling we have chemically synthesized 14 fragment analogs of residues 28 to 38 of horse cytochrome c as described in the previous year. The binding constants of these analogs with a two-fragment complex containing a heme fragment residues 1 to 25 and an apofragment of residues 38 to 104 to form a productive, three-fragment complex was determined as a function of temperature. The results have shown a) replacement of invariant proine 30, leucine 32 or glycine 34 profoundly affects the enthalpy-entropy compensation involved in the binding, resulting in destabilization while the effect of substitution of partially invariant leucine 35 with isoleucine is small; b) hydrophobic interaction is not primarily responsible for the destabilization. The thermal transition of the absorption band at 695 nm and kinetics of this transition or the fragment exchange of the complex have indicated that substitution of some of invariant residues (e.g. leucine 32, glycin 34) would affect not only the interatomic itneractions at the substituted sites but also those involved in ligation of methionine 80 to the heme iron or a concerted global motion. Thus, the invariance of leucine 32 and glycine 34 may be related to the coupling phenomenon. There may also be a link between the enthalpy-entropy compensation phenomenon known for protein folding and the mechanisms of the coupling. Our aim is to find the mechanism of the coupling which underlies the structure, function and folding of proteins.